Method for generating a series of ultra-thin sections using an ultramicrotome, method for three-dimensional reconstruction of a microscopic sample, ultramicrotome system and computer program

Abstract

A method is proposed for generating a series of ultra-thin sections of a microscopic sample (10), wherein the sections (11) are detached from the sample (10) using an ultramicrotome (100) and wherein the sections (11), which are detached from the sample (10) are caused to float on a liquid surface and are thereafter transferred onto a solid carrier element (20). For at least for some of the sections (11) detached from the sample (10) a position and an orientation on the solid carrier element (20) are determined by monitoring the placement of these sections (11) onto the solid carrier element (20) using a monitoring system (400) comprising a camera (410), obtaining monitoring data. A method (2000) for the three-dimensional reconstruction of a microscopic sample (10), a microtome system and a computer program are also part of the present invention.

1 . A method for generating a series of ultra-thin sections of a microscopic sample, the method comprising:
 detaching the sections from the sample using an ultramicrotome wherein the sections detached from the sample are made to float on a liquid surface and thereafter are transferred onto a solid carrier element;   determining, for at least one of the sections detached from the sample, a position and an orientation on the solid carrier element by monitoring the placement of the sections onto the solid carrier element using a monitoring system comprising a camera; and   obtaining monitoring data.   
     
     
         2 . The method according to  claim 1 , wherein the position and the orientation on the solid carrier element determined for at least one of the sections comprises at least one of a rotation, a displacement, a distortion, a folding, and a flip-over of the sections. 
     
     
         3 . The method according to  claim 1 , comprising:
 dissecting, after placing the sections detached from the sample onto the solid carrier element, the solid carrier element into several carrier element parts;   placing the carrier element parts onto a transfer device; and   determining, for at least for one of the carrier element parts generated by dissecting the carrier element, a position and/or an orientation-on the transfer device by monitoring the placement of the carrier element parts onto the transfer device using the same monitoring system or a further monitoring system.   
     
     
         4 . The method according to  claim 1 , wherein the monitoring using the monitoring system comprises acquiring moving-image data, wherein the sections are tracked in the moving-image data. 
     
     
         5 . The method according to  claim 1 , in which the detachment of at least one of the sections from the sample encompasses the generation of section ribbons in which the sections adhere to each other, and in which the sections are placed at least in part in the form of such section ribbons onto the solid carrier element. 
     
     
         6 . The method according to  claim 1 , in which the detachment of at least one of the sections from the sample encompasses the generation of individual sections not adhering to each other, and in which the sections are placed at least in part in the form of such individual sections onto the solid carrier element. 
     
     
         7 . The method according to  claim 1 , in which the monitoring data comprise, or are used in generating a dataset comprising, for at least one of the sections detached from the sample for which the position and the orientation on the solid carrier element is determined, at least one of a position indicator and an orientation indicator relative to a reference position or orientation. 
     
     
         8 . The method according to  claim 1 , wherein the solid carrier element is provided to comprise machine-readable identifiers for target and/or-reference positions of the sections to be placed onto the solid carrier element. 
     
     
         9 . A method for the three-dimensional reconstruction of a microscopic sample, the method comprising:
 microscopically investigating the sections of the sample using a microscopic device   acquiring section image data, wherein the section image data are assembled into a volume image;   generating the sections of the sample by a method according to  claim 1 , wherein the assembling the section image data into the volume image is performed based on the monitoring data obtained using the monitoring system or based on data derived from the monitoring data.   
     
     
         10 . The method according to  claim 9 , wherein the section image data are acquired and/or assembled into the volume image by using an image acquisition and/or analysis-system, the monitoring data or the data derived from the monitoring data being transferred from the monitoring system to the image acquisition and/or analysis-system. 
     
     
         11 . The method according to  claim 9 , further comprising:
 adjusting parameters of the microscopic device based on the monitoring data obtained using the monitoring system or data derived from the monitoring data.   
     
     
         12 . An ultramicrotome system for generating a series of ultra-thin sections of a microscopic sample, the ultramicrotome system being adapted to detach the ultra-thin sections from the sample, to cause the sections to float on a liquid surface, and to thereafter transfer the sections onto a solid carrier element,
 the ultramicrotome system comprising:   a monitoring system comprising a camera, the monitoring system being adapted to:
 determine for at least one of the sections detached from the sample a position and an orientation on the solid carrier element by monitoring a placement of these sections onto the solid carrier element; and 
 obtain monitoring data. 
   
     
     
         13 . The microtome system according to  claim 12 , comprising a feeding system adapted to:
 provide the solid carrier element in the form of a ribbon-like structure; and   position the solid carrier element for placing the sections detached from the sample thereon.   
     
     
         14 . A non-transitory computer-readable medium storing-computer-executable program code for performing the method according to  claim 1  when the computer program is executed by a processor. 
     
     
         15 . The method according to  claim 1 , comprising:
 dissecting, after placing the sections detached from the sample onto the solid carrier element, the solid carrier element into several carrier element parts;   placing the carrier element parts onto a transfer device; and   determining, for at least for one of the carrier element parts generated by dissecting the carrier element, an orientation on the transfer device by monitoring the placement of the carrier element parts onto the transfer device using the same monitoring system or a further monitoring system.   
     
     
         16 . The method according to  claim 1 , wherein the solid carrier element is provided to comprise machine-readable identifiers for target positions of the sections to be placed onto the solid carrier element. 
     
     
         17 . The method according to  claim 9 , wherein the section image data are acquired and/or assembled into the volume image by using an image analysis system, the monitoring data or the data derived from the monitoring data being transferred from the monitoring system to the image analysis system.